The zebrafish has become an ideal model to study blood vessel formation during embryonic development. The transparency and external development of the zebrafish embryo allows detailed and direct observation of blood vessel growth as it occurs in vivo. Furthermore, the zebrafish is amenable to a variety of different genetic manipulations making it possible to assess gene function during vascular development. Despite the success of forward genetic screens in identifying novel genes required for blood vessel formation, these approaches are labor- and time-intensive. Furthermore, due to the size of the vertebrate genome, generation time, and maintenance costs, screening to saturation is difficult. The increasing availability of genomic and expressed sequences has revealed more than 100 candidate genes that are expressed in endothelial cells and are implicated in vascular development, underscoring the need for a definitive reverse genetic approach to determine the function of these genes. Recently, we have successfully applied zinc finger nucleases for targeted gene inactivation in the zebrafish. In this application, we will build on our previous work and apply this technology in the context of a reverse genetic screen to determine the function of candidate genes implicated in blood vessel development. In Aim 1, we will construct high- specificity zinc finger proteins against target sites in more than 30 endothelial cell-expressed genes. These zinc finger proteins will be used to construct zinc finger nucleases (ZFNs) that will be functionally validated in zebrafish embryos. In Aim 2, we will utilize ZFNs to generate founder lines that bear null mutations at targets sites within candidate genes. Aim 3 will focus on the detailed phenotypic characterization of mutant embryos bearing mutations in candidate genes. In particular, we will focus on defects in vascular morphogenesis, differentiation and function. Comparison of defects between mutants, along with previously described mutants, will allow preliminary assembly of these genes into genetic pathways. Subsequent epistasis experiments will allow more definitive genetic characterization of these pathways. This novel application of ZFNs in the context of a reverse genetic screen for genes important during vascular development will provide a framework for similar approaches to dissect other biological processes in the zebrafish. Furthermore, the future widespread access of the ZFN technology applied in this proposal to the zebrafish community will greatly facilitate the generation of collections of new mutant lines.